1. Field of the Invention
This invention relates to an analysis method and apparatus, wherein a light beam is totally reflected from an interface between a thin film layer, which is in contact with a sample, and a dielectric material block, an evanescent wave is thus caused to occur, and an alteration occurring with an intensity of the totally reflected light beam due to the occurrence of the evanescent wave is measured for an analysis of the sample. This invention also relates to an analysis unit for use in the analysis method and apparatus.
2. Description of the Related Art
As analysis apparatuses utilizing evanescent waves, surface plasmon sensors have heretofore been known. In metals, free electrons vibrate collectively, and a compression wave referred to as a plasma wave is thereby produced. The compression wave occurring on the metal surface and having been quantized is referred to as the surface plasmon. With the surface plasmon sensors, characteristics of samples are analyzed by the utilization of a phenomenon, in which the surface plasmon is excited by a light wave. Various types of surface plasmon sensors have heretofore been proposed. As one of well known surface plasmon sensors, a surface plasmon sensor utilizing a system referred to as the Kretschman arrangement may be mentioned. The surface plasmon sensor utilizing the system referred to as the Kretschman arrangement is described in, for example, Japanese Unexamined Patent Publication No. 6(1994)-167443.
Basically, the surface plasmon sensor utilizing the system referred to as the Kretschman arrangement comprises (i) a dielectric material block having, for example, a prism-like shape, (ii) a metal film, which is formed on one surface of the dielectric material block and is brought into contact with a sample, (iii) a light source for producing a light beam, (iv) an optical system for irradiating the light beam to the dielectric material block at various different incidence angles such that a total reflection condition may be obtained at an interface between the dielectric material block and the metal film, (v) a photo detecting means for detecting the intensity of the light beam, which has been totally reflected from the interface described above, and (vi) analysis means for analyzing the state of surface plasmon resonance in accordance with the result of the detection having been made by the photo detecting means.
In order for the various different incidence angles described above to be obtained, a light beam having a comparatively small beam diameter may be caused to impinge upon the aforesaid interface with the incidence angle being altered. Alternatively, a light beam having a comparatively large beam diameter may be caused to impinge upon the aforesaid interface in a state of converged light or in a state of a divergent light, such that the light beam may contain components, which impinge at various different incidence angles upon the interface. In the former case, the reflected light beam, which is reflected from the interface with its reflection angle altering in accordance with the alteration of the incidence angle of the incident light beam, may be detected with a small photodetector, which moves by being interlocked with the alteration of the reflection angle, or may be detected with an area sensor extending in the direction of alteration of the reflection angle. In the latter case, the light beam may be detected with an area sensor extending in a direction such that the area sensor is capable of receiving all of the light beam components having been reflected from the interface at various different reflection angles.
With the surface plasmon sensor having the constitution described above, in cases where the light beam impinges at a specific incidence angle θSP, which is not smaller than the total reflection angle, upon the metal film, an evanescent wave having an electric field distribution occurs in the sample, which is in contact with the metal film, and the surface plasmon is excited by the evanescent wave and at the interface between the metal film and the sample. In cases where the wave vector of the evanescent wave coincides with the wave vector of the surface plasmon, and wave number matching is thus obtained, the evanescent wave and the surface plasmon resonate, and energy of the light transfers to the surface plasmon. As a result, the intensity of the reflected light beam, which is totally reflected from the interface between the dielectric material block and the metal film, becomes markedly low. Ordinarily, the lowering of the intensity of the reflected light beam is detected as a dark line by the photo detecting means described above.
The resonance described above occurs only in cases where the incident light beam is P-polarized light. Therefore, it is necessary for the incident light beam to be set previously so as to impinge upon the aforesaid metal film as the P-polarized light.
The specific incidence angle θSP, which is not smaller than the total reflection angle, and which is associated with the lowering of the intensity of the reflected light beam, will hereinbelow be referred to as the attenuated total reflection angle (ATR angle) θSP. In cases where the wave number of the surface plasmon is found from the ATR angle θSP, a dielectric constant of the sample is capable of being calculated. Specifically, the formula shown below obtains.
            K      SP        ⁡          (      ω      )        =            ω      c        ⁢                                                      ɛ              m                        ⁡                          (              ω              )                                ⁢                      ɛ            s                                                              ɛ              m                        ⁡                          (              ω              )                                +                      ɛ            s                              wherein KSP represents the wave number of the surface plasmon, ω represents the angular frequency of the surface plasmon, c represents the light velocity in a vacuum, ∈m represents the dielectric constant of the metal, and ∈s represents the dielectric constant of the sample.
Specifically, in cases where the dielectric constant ∈s of the sample is found, the refractive index of the sample, or the like, is capable of being found in accordance with a predetermined calibration curve, or the like. Therefore, in cases where the ATR angle θSP is found, the dielectric constant ∈s of the sample is capable of being calculated. Accordingly, the characteristics with regard to the refractive index of the sample are capable of being calculated.
As a similar sensor utilizing the evanescent wave, a leaky mode sensor has heretofore been known. (The leaky mode sensor is described in, for example, “Surface Refracto-sensor using Evanescent Waves: Principles and Instrumentations” Takayuki Okamoto, Bunko Kenkyu, Vol. 47, No. 1, 1998.) Basically, the leaky mode sensor comprises (i) a dielectric material block having, for example, a prism-like shape, (ii) a cladding layer, which is formed on one surface of the dielectric material block, (iii) an optical waveguide layer, which is formed on the cladding layer and is brought into contact with a sample, (iv) a light source for producing a light beam, (v) an optical system for irradiating the light beam to the dielectric material block at various different incidence angles such that a total reflection condition may be obtained at an interface between the dielectric material block and the cladding layer, (vi) photo detecting means for detecting the intensity of the light beam, which has been totally reflected from the interface described above, and (vii) analysis means for analyzing the state of excitation of a guided mode in accordance with the result of the detection having been made by the photo detecting means.
With the leaky mode sensor having the constitution described above, in cases where the light beam impinges at an incidence angle, which is not smaller than the total reflection angle, upon the cladding layer via the dielectric material block, only the light having a certain specific wave number, which light has impinged at a specific incidence angle upon the cladding layer, is propagated in the guided mode in the optical waveguide layer after passing through the cladding layer. In cases where the guided mode is thus excited, approximately all of the incident light is taken into the optical waveguide layer. Therefore, in such cases, the attenuated total reflection occurs, and the intensity of the light totally reflected from the aforesaid interface becomes markedly low. Also, the wave number of the guided optical wave depends upon the refractive index of the sample, which is located on the optical waveguide layer. Therefore, incases where the ATR angle θSP is detected, the refractive index of the sample and characteristics of the sample with regard to the refractive index of the sample are capable of being analyzed.
In the fields of pharmaceutical research, and the like, the surface plasmon sensor and the leaky mode sensor described above are often utilized for random screening for finding out a specific substance, which is capable of undergoing the binding with a desired sensing substance. In such cases, the sensing substance is fixed to the aforesaid thin film layer (the metal film in the cases of the surface plasmon sensor, or the combination of the cladding layer and the optical waveguide layer in the cases of the leaky mode sensor), and a liquid (a liquid sample) containing a test body is introduced on the sensing substance. Also, at each of stages after the passage of predetermined periods of time, the aforesaid ATR angle θSP is measured.
In cases where the test body contained in the liquid sample is a substance capable of undergoing the binding with the sensing substance, the refractive index of the sensing substance alters with the passage of time. Therefore, the aforesaid ATR angle θSP is measured at each of stages after the passage of predetermined periods of time, and a judgment is made as to whether an alteration of the ATR angle θSP has been or has not been occurred. In this manner, a judgment is capable of being made as to whether the binding of the test body with the sensing substance has or has not occurred, i.e. as to whether the test body is or is not the specific substance capable of undergoing the binding with the sensing substance. Examples of the combinations of the specific substances and the sensing substances include the combination of an antigen and an antibody and the combination of an antibody and a different antibody. Specifically, examples of the analyses with regard to the combinations of the specific substances and the sensing substances include an analysis, wherein a rabbit anti-human IgG antibody is employed as the sensing substance, a detection is made as to whether a human IgG antibody acting as the test body has or has not been bound to the rabbit anti-human IgG antibody, and a quantitative analysis of the human IgG antibody is made.
In order for the state of the binding of the test body, which is contained in the liquid sample, with the sensing substance to be detected, the ATR angle θSP itself need not necessarily be detected. Alternatively, for example, the liquid sample containing the test body may be introduced on the sensing substance, and thereafter the quantity of the alteration of the ATR angle θSP may be measured. Also, the state of the binding of the test body with the sensing substance may be detected in accordance with the quantity of the alteration of the ATR angle θSP.
Also, the inventors proposed a sensor for detecting the state of the attenuated total reflection by use of an analysis chip, which has a well-like shape and is easy to process. (The proposed sensor is described in, for example, U.S. Pat. No. 6,597,456.) With the analysis chip having the well-like shape, in cases where, for example, a liquid sample is used as the sample, only a small amount of the liquid sample to be introduced into the analysis chip may be prepared for the analysis. Further, in cases where a table capable of supporting a plurality of analysis chips is utilized, analyses of various kinds of samples are capable of being made quickly and easily.
Furthermore, as the analysis apparatus of the type described above, there has been known a sensor for making an analysis, wherein a liquid sample is continuously supplied by use of a flow path means and onto a planar analysis chip, to which a sensing substance has been fixed. In cases where the sensor provided with the flow path means is utilized, during the detection of the state of the binding of the sensing substance and a specific substance with each other, the fresh liquid sample is continuously supplied onto the analysis chip. Therefore, the concentration of the test body contained in the liquid sample supplied onto the analysis chip does not alter, and the detection of the state of the binding is capable of being made accurately. Also, after it has been detected that the sensing substance and the specific substance have been bound to each other, a liquid sample containing no specific substance may be caused to flow on the analysis chip, to which the product of the binding of the sensing substance and the specific substance with each other has been fixed. In this manner, the state of separation of the sensing substance and the specific substance from each other is capable of being detected. Further, in cases where, for example, a gas is employed as the sample, or in cases where a liquid sample containing a gas is employed as the sample, the sample is capable of being easily supplied onto the analysis chip by use of the flow path means.
Also, recently, a wide variety of operations for detecting various kinds of reactions are performed, and various kinds of solvents for samples are utilized. Examples of the solvents include the solvents, such as water, which comparatively readily evaporate. The evaporation of water acting as the solvent results in an alteration of the refractive index of the sample and an alteration of the detection signal. In such cases, an accurate analysis is not capable of being made. In such cases, by the provision of the flow path means described above, the evaporation of the sample is capable of being suppressed, and a reliable detection signal is capable of being obtained.
As described above, by the provision of the flow path means, various effects are capable of being obtained. However, with the sensor provided with the flow path means, the problems occur in that, in order for the sample to be supplied continuously onto the analysis chip, it is necessary for a large amount of the sample to be prepared. Also, the problems occur in that the analyses of various kinds of samples are not capable of being made quickly.
As for the metal film, on which the surface plasmon resonance occurs, it is necessary for various kinds of proteins to be fixed to the metal film in accordance with the kinds of the reactions to be detected. However, ordinarily, the liquids containing the proteins are expensive. Therefore, it is desired that the liquids containing the proteins are capable of being utilized iterately for the fixation of the proteins in the plurality of the wells.
In cases where the top surface of the metal film is widely open, a liquid containing a protein is capable of being supplied by use of an ordinarily utilized pipette, or the like, and then sucked up after a predetermined period of time necessary for the fixation has elapsed. (In certain cases, in order for the fixation to be promoted, the suction and the discharging of the liquid containing the protein may be iterated at the site.) The liquid containing the protein is thus capable of being recovered. Also, the recovered liquid containing the protein is then capable of being utilized for the fixation of the protein in a different well. In order for the protein fixation in the plurality of the well to be performed efficiently and easily, the top surface of the metal film should preferably be capable of being set to be widely open.
Further, as for certain kinds of analyses, it will be desired that the liquid sample is capable of being supplied onto the metal film by use of a flow path. Also, as for different kinds of analyses, it will be desired that, instead of the flow path being located, the liquid sample is capable of being supplied directly into the well. Therefore, it is desired that the analysis apparatus enables the selection of the supply of the liquid sample by use of the flow path or the supply of the liquid sample without the flow path being used.
Furthermore, there has been known a technique, wherein a reference signal is utilized such that errors in analysis results due to bulk effects, temperature changes, fluctuations of the light source, or the like, may be eliminated, and the analysis accuracy is thereby enhanced. In such cases, it is necessary that two kinds of analysis chips, i.e. an analysis chip for an analysis of a sample and an analysis chip for reference, be prepared.
Also, the analysis apparatuses, such as the surface plasmon sensors or the leaky mode sensors, are required to have a fine angle accuracy. Therefore, besides the reference analysis, it is desired that two kinds of analyses be capable of being made simultaneously in one analysis chip, in which the measurement conditions are kept approximately identical, at the time of experiments for comparison, and the like.